The present invention relates generally to protein chemistry of granulocyte-specific colony stimulating factors (G-CSF), and more particularly to a process for purifying G-CSF by selective precipitation.
The differentiation and proliferation of mammalian hematopoietic cells is regulated by secreted glycoproteins collectively referred to as colony stimulating factors (CSFs). Among these colony stimulating factors are G-CSFs, which induce differentiation and expansion of granulocyte-committed progenitor cells from multipotent hematopoietic stem cells. When administered to mammals, G-CSFs promote a dramatic increase in circulating granulocyte populations. Both murine and human G-CSFs have been isolated and partially characterized.
Due to potential clinical utility as a stimulator of granulocytic cell precursors, there is considerable interest in G-CSF. Therapeutic compositions with G-CSF activity could be employed to potentiate immune responsiveness to infectious pathogens, or to assist in reconstituting normal blood cell populations following radiation or chemotherapy-induced hematopoietic cell suppression. G-CSF may also find application in the treatment of certain leukemias, due to its ability to cause differentiation of certain neoplastic cells of hematopoietic lineage.
In order to fully characterize the biological activities of G-CSFs, methods have been developed to obtain protein from culture supernatants of cells known to constitutively produce G-CSF. For example, Watson et al. (J. Immunol. 137:854, 1986) reported a procedure for purifying human G-CSF produced from a 5637 bladder carcinoma. Twenty liters of 5637-conditioned medium was concentrated by 80% ammonium sulfate precipitation, and the precipitate dialyzed against phosphate-buffered saline (PBS) and fractionated on a gel filtration column at 4.degree. C. Active fractions containing protein were pooled and subjected to three sequential reversed-phase high performance liquid chromatography (HPLC) steps. Nomura et al. (EMBO J. 5:871, 1986) reported a method for purifying human G-CSF produced from a human oral cavity carcinoma cell line (CHU-2), known to constitutively produce G-CSF. Serum-free CHU-2 conditioned medium was concentrated to 5 ml by ultrafiltration, which was further concentrated by applying to an Ultrogel AcA-54 gel filtration column and eluting to obtain two active fractions. Half of one fraction (containing 28 mg protein) was subjected to reversed-phase HPLC and gel-permeation HPLC to yield 140 .mu.g protein.
Recombinant human G-CSF (rhG-CSF) has also been produced by expressing a rhG-CSF gene using an expression vector promoting secretory expression of G-CSF from transformed host cells, thus enabling production of larger quantities of hG-CSF. Although hG-CSF can be expressed in high levels in recombinant hosts, the resulting recombinant protein must be purified to homogeneity to enable clinical use. For example, Nagata et al. (Nature 319:415, 1986 and EMBO J. 5:575, 1986) reported the isolation of two cDNAs from the CHU-2 cell line which encode G-CSF protein. European Patent Application Serial Nos. 0 215 126 to Ono et al. and 0 220 520 to Yamazaki et al. disclose additional details concerning the cloning and expression of hG-CSF. Ono et al. specifically disclosed expression of CHU-2-derived G-CSF in E. coli. Recombinant hG-CSF was reported to have been purified by first lysing transformants to obtain cell supernatants. The supernatants were concentrated by gel filtration on an Ultrogel AcA-54 column, further concentrated with an ultrafiltration apparatus, and purified by two sequential purifications by adsorption on a reversed-phase C18 column. rhG-CSF was purified from the resulting fractions by HPLC based on molecular size and by SDS-polyacrylamide gel electrophoresis.
Copending U.S. patent application Ser. No. 7/029,742 describes recombinant hG-CSF analog proteins produced by DNA having alterations which enhance expression of rhG-CSF in yeast systems. These rhG-CSF proteins were initially purified from yeast supernatants by the multiple step methods of Watson et al. (discussed above). Significant yield losses were realized in the HPLC steps, however, because G-CSF adheres to the surfaces of glassware used in the purification process.
In an effort to overcome the aforementioned problems associated with multiple step purification methods, alternative methods of purification have been attempted, for example, precipitation of hG-CSF by addition of ammonium sulfate salt. Although hG-CSF does precipitate from aqueous solutions upon addition of ammonium sulfate salt, other proteins precipitate simultaneously. Ammonium sulfate salt does not therefore selectively purify hG-CSF.
The various purification methods described above each require multiple steps in order to achieve acceptable levels of purity. On a commercial scale, combining various steps is both impractical and uneconomical because each purification step not only adds to the cost of the final product but also causes successive yield losses of hG-CSF protein. Yield losses of hG-CSF are also caused by the tendency of G-CSF protein to adhere to the surfaces of glassware used in each step of the purification process. Thus, in order to purify large quantities of rhG-CSF economically, highly selective and simplified methods requiring fewer steps and providing higher yields of active protein are desirable.